Optical substrate for enhanced detectability of fluorescence

ABSTRACT

A sample substrate adapted for use with fluorescence excitation light with a first wavelength. A reflector is disposed on a base. The reflector includes a reflecting multilayer interference coating with at least two layers. Not all of the layers L fulfill a quarterwave condition: dL·nL=(2N+1)·¼ wherein dL is a physical thickness of layer L, nL is an index of refraction of layer L at the first wavelength, N is an integer equal to or greater than zero and 1 is the first wavelength. Thicknesses of the layers ensure that any fluorescent sample material disposed on top of said multilayer interference coating would be located near an antinode of a standing wave formed by the excitation light with the first wavelength incident on said substrate.

BACKGROUND OF THE INVENTION

The present invention relates to sample substrates, such as plates,slides and cells, for use in examining, indicating, analyzing oridentifying fluorescent, phosphorescent or luminescent sample materials,e.g. tagged molecular biological specimens, and in particular relates tosuch sample holders whose optical structures are adapted for enhancingfluorescence detection and imaging. The format of the sample substratesof the present invention can be adapted to formats typically used inthis field, such as for example the standard format of microtiterplates.

Fluorescence microscopy is often used in the fields of molecularbiology, biochemistry and other life sciences for analyzing biologicalmolecules, including nucleic acids (DNA, RNA) and proteins (enzymes,antigens, etc.) that have been tagged or labeled with fluorescentprobes. One such use is DNA diagnostics, such as for gene detection, inwhich a DNA sample is deposited on and bound to a glass substrate. Thebound DNA on the substrate can then be imaged by fluorescence. Thefluorescence of a sample was originally assessed by visual inspectionthrough a conventional microscope, but this manual method has provedtime-consuming and costly. Many different high-speed automatedfluorescence imaging systems are now available.

An important figure of merit for fluorescence detection and measurementinstruments is sensitivity, which is primarily determined by thesignal-to-noise ratio (SNR) of the optical imaging system of theinstrument. A well-designed imaging system has a signal-to-noise ratiothat is limited by its light collection ability and not by internalnoise sources. The theoretical SNR of such a system is expressed interms of the number of photoelectrons at the cathode when using aphotomultiplier tube (PMT), which in turn essentially depends upon thenumber of photons that reach the detector from the area of interest onthe sample substrate, the quantum efficiency of the detector, and thenumber of dark electrons generated by the detector.

One obvious approach to increasing SNR, and thereby improvingsensitivity, is to reduce background noise. Sources of background noiseinclude specular or diffuse reflection of the fluorescence-stimulatinglaser light from the sample, autofluorescence of the substrate holdingthe sample, autofluorescence from the optics in the light path of theoptical imaging system, stray light, and dark current of the detector.Stray light reaching the detector can be significantly reduced by propersize and placement of apertures in the imaging system. Both stray lightand much of the reflected laser light can be rejected, while passing thefluorescent light, by using dichroic and other spectral filters andbeamsplitters in the system. Autofluorescence of the optical elementscan be reduced by avoiding use of lens cements in the light path, usingglass instead of polymeric lenses, or using curved mirrors instead oflenses wherever possible.

Autofluorescence of the substrate can be reduced by using lowfluorescence materials, such as an ultrathin or opaque glass substrate.For example, in U.S. Pat. No. 5,095,213 Strongin discloses a plasticslide that is rendered opaque and substantially nonfluorescent with aquantity of black carbon powder in the plastic. Another way of handlingautofluorescence is to use a pulsed or modulated excitation and to takeadvantage of the differences in emission decay rates between backgroundfluorescence and specimen fluorescence, as disclosed in U.S. Pat. No.4,877,965 to Dandliker et al. and U.S. Pat. No. 5,091,653 to Creager etal.

In U.S. Pat. No. 5,552,272, Bogart discloses an assay system and methodfor detecting the presence or amount of an analyte of interest. Itincludes a test substrate with an optically active surface that enhancesthe color contrast, i.e. differences in the observed wavelength (orcombination of wavelengths) of light from the surface, between thepresence and absence of the analyte in a sample applied onto the testsubstrate. In particular, the substrate may comprise a reflective solidoptical base, such as a silicon wafer or metallic (e.g., aluminum) base,with an optical thin film coating thereon. The coating may compriseseveral layers, including for example an attachment layer on the uppersurface of the base, and a receptive layer on the upper surface of theattachment layer containing a specific binding partner for the analyteof interest. The total coating thickness is selected to cause incidentlight to undergo thin film interference upon reflection, such that aspecific color is produced. Specifically, the coating material(s) shouldhave an overall thickness of a quarterwave of the unwanted color to beattenuated so that destructive interference of that color will occur.The substrate therefore has a particular background color, which canthen be used as a comparative reference against a different observedcolor when an analyte of interest is present. Both qualitative visualinspection and quantitative instrumented measurement are suggested.Polarization contrast by means of an ellipsometer is also suggested.

One example of the use to which the Bogart invention has been put byBiostar, Inc. of Boulder, Colo., the assignee of the aforementionedpatent, is an optical immunoassay (OIA) diagnostic screening test forthe rapid detection (in under 30 minutes) of the presence of specificantigens of infectious pathogens in a sample taken from a patient.Commercial products include test kits for group A and group Bstreptococci and for chlamydia trachomatis. These particular assays aregiven as examples in the Bogart patent, are described in package insertsfor the corresponding Biostar products and are also described in anumber of published articles in medical journals. Briefly, they all relyon direct visual detection of a change in the color of light reflectionoff of the test substrate due to a physical change in the opticalthickness of a molecular thin film coating on the substrate surfacewhich results from binding reactions between an immobilized antibody onthe test surface and a specific antigen that may be present in a drop ofsample liquid applied to the test surface. The original bare testsurface has a thin film thickness that results in a predominant visualbackground gold color when white light is reflected off of the surface.The antigen-antibody binding reaction that occurs when the specificantigen of interest is present in the applied sample results in anincrease in the thin film thickness that causes a corresponding changein the color of the test surface from gold to purple. If on the otherhand, the antigen is not present in the sample, no binding takes place,the original thin film thickness remains unchanged and the test surfaceretains its original gold color, indicating a negative result. Thisdiagnostic assay tool is very sensitive and easily interpreted.

Bogart further discloses, in another embodiment of his invention (FIG.17 of the aforementioned patent), the use of these substrates forenhanced fluorescence detection. After the analyte of interest has beenbound to the surface by reaction with the specific binding partner inthe receptive layer of the substrate coating, fluorescent labelmolecules may be attached to the analyte. In particular, the fluorescentmolecules may be attached to any suitably selective and specificreceptive material or reagent, such as a secondary antibody, and appliedto the surface. The fluorescent labels are thus bound to the analyte ofinterest on the surface, if present, and immobilized to the surfacethrough the analyte bridge. Directing light of an excitation wavelengthonto the surface stimulates fluorescence of any of the label bound tothe surface, thereby revealing the presence of the analyte of interest.Because the maximum fluorescence wavelength may not be shifted farenough from the excitation wavelength to be distinguished, thereflective substrate may have an antireflection layer whose thickness isselected to suppress reflection of the excitation wavelength, therebyreducing the background noise reaching the detector. Bogart states thatthe fluorescent signal generation is not dependent on the filmthickness. Though the fluorescence signal S might be increased byincreasing the output power of the laser, reflected laser noise willalso increase, with possibly little improvement in the resulting SNR.

In U.S. Pat. No. 6,008,892 Kain et al disclose a sample substrate whichis reflective for the excitation wavelength. This substrate has atransparent coating layer thereon with controlled thickness that hasbeen selected to ensure that a molecular sample placed on top of thecoating layer is located at an antinode for the excitation light. Inparticular, the substrate includes a rigid base with a specularlyreflective upper surface. The transparent coating on the upper surfaceof the base has a thickness selected such that for a particularexcitation wavelength of light at normal incidence, the optical pathfrom the top of the coating to the base reflecting surface issubstantially an odd multiple (1, 3, 5, etc.) of one-quarter wavelengthof the excitation light. The optical path length of the material isdefined by the wavelength of light, the index of refraction of thematerial, and the angle of propagation through the material. In thereflective sample substrate the reflecting surface of the base is at awell defined depth slightly below the physical surface of the base by anamount equal to the sum of the skin (or penetration) depth of thereflective surface material and the optical depth of any surfaceoxidation on the base. By placing the sample on the coating layer at ornear the antinode of the excitation light, maximum fluorescenceexcitation occurs. A reflective substrate also enhances fluorescencecollection by nearly doubling the solid collection angle of afluorescence imaging microscope system. Thus, the total fluorescencesignal is increased, leading to a much improved signal-to-noise ratio.Also, because the coating layer is very thin, there is reducedfluorescence background noise from this material.

As Kain et al describe the base can be made completely of metal or maybe composed of a rigid bottom layer with a top metal coating. The metalcan be aluminum, silver, gold or rhodium. The transparent coating may bea single layer of dielectric material, such as silica, alumina or afluoride material (such as MgF₂). Alternatively, the transparent coatingcould be a multilayer coating with the top layer being a chemicallyreactive material for binding a specified biological sample constituentthereto.

Kains concept of applying a transparent quarterwave layer on areflecting surface is limited to reflectors with rigid surfaces. Herelight is not penetrating beneath the physical surface of the reflectoror the penetration is limited to at most some nanometers beneath thesurface (skin depth). Practically this represents a limitation tometallic surfaces such as metal substrates or metallic coatings.

As soon as dielectric layers significantly contribute to the reflection,it is difficult to define a penetration depth and the concept of addinga quarterwave layer fails. For example for aluminum mirrors very oftenadditional dielectric layers are used to enhance the reflectivity of themetal which results in so called “pumped metallic mirrors”. In this casethe dielectric layers are part of the mirror and the last dielectriclayer forms the physical surface of the mirror. Light penetrates intosuch a mirror stack and interference effects together with the metallicreflectivity establish the optical characteristics. Adding an additionalquarterwave layer or an odd multiple to such a system very often failsto produce an antinode at the location of fluorescent sample when placedon this layer. In addition in most cases the interference system isdisturbed which might result in a drastic decrease of reflectivity.

However as long as the odd multiple quarterwave condition is fulfilledwith respect to the metallic surface it is even possible to enhancefluorescence using the reflectivitiy contribution of the quarterwavelayers. Chaton et al give one example in WO 02/48691 where this effectcan be seen. Chaton et al describe the use of dielectric quarterwavestacks which have a mirror function on a silicon substrate in order toenhance the fluorescence. The reflectivity of silicium at 550 nm is atabout 42%. This results in a square electromagnetic field amplitude (E²)on the metallic surface of about 10% of the square of the fieldamplitude of the free propagating wave E²(PW). Applying a singlequaterwave layer of SiO₂ on that rigid metallic surface (following oneembodiment of the invention of Kain et al) results in a field amplitudewhich is 25% higher than E²(PW) therefore resulting in an enhancementfactor of 1.25. Chaton et al use a quarterwave stack with a designwavelength of 550 nm in order to reinforce reflectivity. This means thata system of alternating high and low index dielectric layers is used toincrease the reflectivity, where each individual layer has an opticalthickness of one quarter of the design wavelength. As an example Chatonet al use a five layer system on silicon with SiO₂ (94 nm layerthickness, 3 layers) and Si₃N₄ (69 nm layer thickness, 2 layers) ascoating materials. This results in a reflectivity of slightly below 60%.Mirrors based on quarterwave stacks are known as Bragg mirrors.Typically such a Bragg mirror is based on a quarterwave stack where theoutermost layer is a high index layer. However Chaton et al use asoutermost quarterwave layer an SiO₂ layer, which is the low indexmaterial. It is known that the outermost layer being a low index layerresults in lower reflection values. Removing the outermost layer (lowindex layer) would even result in higher reflectivity. As the Chaton etal describe they use SiO₂ as outermost layer in order to create aphysical surface which is compatible with the linker chemistry. Howeverthere is one additional positive effect the authors did not mention: Ourinvestigations showed that using a low index layer as outermost layerresults in a field amplitude for 550 nm on the surface which isfortunately maximum, 220% more than E²(PW) resulting in an enhancementfactor of 2.2. If the authors had used a high index layer as outermostlayer the electromagnetic field amplitude would have been minimum.

With a coating design as used by Chaton et al the reflection band iscentered at the design wavelength of 550 nm and is 200 nm broad. Withinthe reflection band the reflectivity is increased to slightly below 60%.Because Chaton et al did not take into account the antinode condition,they conclude that the quarterwave stack is effectively enhancing thefluorescence signal for wavelengths between 450 nm to 650 nm. Thiswavelength range comprises the wavelengths of the fluorescence materialstypically used for fluorescence lables such as CY3 and CY5.Unfortunately only for the design wavelength the antinode condition iswell fulfilled. For an excitation wavelength much different from 550 nmthis condition is not fulfilled. For 450 nm the enhancement factor is aslow as 0.2 and for 650 nm the enhancement factor decreases down to 1.2.

It is therefore still a problem and would be desirable to create asample which provides optimum enhancement for more than one excitationwavelength well separated from each other. Especially for the excitationwavelengths around 532 nm-548 nm (Cy3) and around 633 nm (Cy5) it isstill an open question how to realize such a sample substrate.

Typically there is an inherent difference of signal intensity for Cy3and Cy5 related for example to differences in affinity of the linkerchemistry, resulting in different signal intensities. Therefore thequestion of how to realize for two or more excitation wavelengthsapproximately the same degree signal intensities may be important. It isan interesting, but even more general aspect to consider the possibilityto adjust the enhancement factors for two or more excitation wavelengthsindependently.

In addition as discussed, the prior art solutions involve in generalinterfaces to metallic layers or substrates. Chaton for examplerestricts the discussion to a silicon substrate. Examples for rigidreflecting surfaces in Kains disclosure always involve metallicsurfaces. It is therefore still a problem and would be desirable tocreate a substrate sample providing enhanced fluorescence signal withoutuse of a metallic interface as do Kain et al as well as Chaton et al.

BRIEF SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide animproved sample substrate which provides increased sample excitation andfluorescence emission without the disadvantage to base the reflection ofthe excitation wavelength completely on the reflectivity of a metalsurface. It is another important aspect of the present invention todisclose a sample substrate providing maximum enhancement of thefluorescence signal for two or more different excitation wavelengths. Itis yet another aspect of the present invention to disclose a samplesubstrate providing application adjusted enhancement factors for two ormore excitiation wavelengths.

The object has been met with a reflective sample substrate comprising aninterference reflector based on a dielectric multilayer coating.Theexcitation light is reflected with 100% or close to 100%. The resultingelectromagnetic field distribution connected to the excitationwavelength outside the sample substrate in the cover region is thereforea standing wave or close to a standing wave with planes of nodes andplanes of antinodes. In the present invention the layers of theinterference reflector have an optimized thickness distribution that hasbeen selected to ensure that an areal fluorescent sample placed on topof the top coating layer is located in the plane of an antinode of theexcitation light.

In this case no quarterwave-thickness requirement for the dielectriclayers have to be fulfilled. This gives an additional degree of freedomfor the design and the maximum or the desired electromagnetic field canbe realized on the physical surface even for two or multiple excitationwavelengths. To be more specific, skipping the condition of usingquarterwave layers or odd multiples of quarterwave dielectric layersleads to an additional degree of freedom in the design which can be usedto fulfill antinode conditions for two or more excitation wavelengthswithout the need for compromise as is the case for prior art solutions.This degree of freedom can be used for samples where metallic reflectinginterfaces are involved as well as for pure dielectric reflectorsleading to which are until now not we did not find in prior artsolutions.

The electrical field distribution within such an interference reflectoris very different from the field distribution known from specularreflection of a metal surface. An additional layer added on top of theinterference reflector can cause a decrease in reflectance. In additionfor such an interference reflector it is not possible to define thelocation of a reflection surface since most of coating layers contributeto the reflection.

However we found during our studies and experiments that, despite thedifficulty to define a reflection surface, it is possible to design aninterference reflector in such a way, that there is a maximum field atthe physical surface of the reflective sample substrate where the arealfluorescent sample is placed. The design can be found with the help ofstandard thin film optimization tools, slightly modified to optimize formaximum reflectivity together with maximum field amplitude at thesurface of the reflective sample substrate.

In this case no additional transparent layer is required, noquarterwave-thickness requirement for the dielectric layers have to befulfilled, so the maximum field can be realized even for two or multipleexcitation wavelengths. In addition such a system can preferably berealized without the use of metal layers or a metal substrate at all.

As already mention such systems can be found using statisticaltechniques as used typically for optical thin film design, modified totake into account as a part of the optimization target the antinodecondition on the physical surface. However we investigated theelectromagnetic field distribution within the substrate samples we hadoptimized with the help of such an optimization procedure. Weinvestigated the sample substrates providing enhanced maximumelectromagnetic field intensity at the physical surface of the samplefor various designs including prior art solutions as disclosed by Kainet al as well as Chaton et al. We found that the electromagnetic fielddistribution provides a minimum which is located one quarterwave belowthe physical surface. This was independent how deep actually theelectromagnetic field propagates into the samples. As a conclusion wethink that the generalization to Kains quarterwave rule is that theelectromagnetic field needs to provide a minimum which is locatedapproximately one quarterwave beneath the physical surface. Thisreflects one aspect of the present invention: As long as the substratesample provides a minimum in the electromagnetic field distributionlocated approximately one quarterwave beneath the physical surface theeletromagnetic field on the physical surface will be enhanced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of a fluorescence imaging system astypically used.

FIG. 2 is a close up side section view of a sample substrate of thepresent invention.

FIG. 3 is a graph showing the normalized square of the electrical fielddistribution (E²)/I₀ ² versus distance from the base of the samplesubstrate shown in FIG. 2. In addition shown is the distribution of theindex of refraction of the multilayer coating.

DETAILED DESCRIPTION OF THE INVENTION

The sample substrate of the present invention can be used in any of awide number of possible fluorescence microscope systems, including, forexample, those described in U.S. Pat. No. 4,284,897 to Sawamura et al.,U.S. Pat. No. 5,091,652 to Mathies et al., U.S. Pat. No. 5,296,700 toKumagai, U.S. Pat. No. 5,381,224 to Dixon et al., and U.S. Pat. No.5,504,336 to Noguchi, as well as U.S. patent application Ser. Nos.08/595,355, 08/616,174 and 08/791,684.

A preferred fluorescence imaging system 1 for use with the presentinvention is illustrated in FIG. 1: A light source 10, for example alaser, produces an stimulating beam 11. The stimulating beam 11 ispreferably a collimated beam of monochromatic coherent light. However, anoncoherent source, such as a light emitting diode (LED) could be usedand a noncollimated source could be coupled to collimating optics tocreate a collimated beam. If the stimulating beam 11 is notmonochromatic, it may be directed through a filter to reduce anyunwanted wavelengths.

The stimulating beam 11 is then directed through lens systems 12, 12′and a beam splitter 15 onto the surface of the sample substrate 20. Anyscanning mechanism that produces a two-dimensional scan may be used tomove the substrate along orthogonal axis in plane with the surface ofthe sample substrate.

The lens system 12 provides coaxial illumination of the sample substratewith the stimulating beam 11. The stimulating beam 11 is an excitationbeam that stimulates fluorescent light emission from the samplesubstrate 20 at the illuminated spot: If there are areal fluorescentsamples 23, 23′ on the illuminated surface of the sample substratestimulation of detectable fluorescent light results. The lens system 12provides as well coaxial collection of the resulting fluorescent lightand a fluorescent beam 29 is formed. To maximize collection efficiency,it is preferred that the lens system 12 has a large numerical aperture.The fluorescent light is then collected by the lens system 12, acting asa condenser, and directed as a retro-beam back along the incident lightpath (but in the opposite direction). Since the fluorescent lightgenerally consists of a broad band of wavelengths different from thewavelength(s) of the incident stimulating beam, and since the systemshould be designed to work with a variety of fluorochromes, the systemis preferably largely achromatic and provides correction of chromaticaberrations over a range of wavelengths. Light passing through the lenssystem 12 impinges upon a photodetector 31, such as a photomultipliertube (PMT).

Whichever imaging system is used, it should preferably be capable ofscanning at high speed over a large scan field with high resolutionimaging and minimal optical aberrations. It should provide coaxialillumination and collection with high collection efficiency. Anachromatic system with excellent color correction, as well as a systemdesigned for minimizing background noise (including autofluorescence) ispreferred.

With reference to FIG. 2, a sample substrate 41 of the present inventioncomprises a base 43 and an interference reflector 45 on top of said base43. The areal fluorescent samples 23, 23′ are applied on top of saidinterference reflector 45.

The base 43 could be made of any material that can be coated. Forexample the base material can be transparent such glass or plastic or itcan be metallic such as aluminum. The use of any other rigid bottomlayer is possible.

The interference reflector 45 comprises a dielectric multilayer coatingwith at least one high index coating layer 47 and at least one low indexcoating layer 49.

As coating materials for example Nb₂O₅, SiO₂, aluminum oxide, magnesiumoxide, oxides of the groups VIb, Vb; IVb, as well as scandium, yttrium,calcium, strontium, zinc, iron, indium, tin, cerium, holmium, as well asoxide of mixtures or alloys of these elements and further oxynitrides ofthe elements Ti, Ta, Zr, Si, Hf, Al, as well as fluorides of theelements Magnesium, Barium, Strontium, Calcium, the rare earths and leadcan be used.

The layers may be coated for example by one of the following methods:thermal and/or electron beam vapor deposition, replication, transfer,film deposition, by processes of the CVD type (LPCVD, PECVD etc.) or ofthe PVD type such as sputtering, i.e. DC magnetron sputtering. Ionassisted deposition processes can be used as well as the sol-gelprocess. The layers may transferred onto the base by one of thefollowing methods: bonding and molecular adhesion.

The top layer of the interference reflector 45 is not necessarily aninert material, but could be biologically active so as to bind with thesample material or a particular constituent of the sample.

In order to maximize the fluorescent emission by maximizing theelectrical field at the location of the areal fluorescent sample, thelayer thickness distribution of the multilayer coating comprised in theinterference reflector 45 is to be optimized.

The optimization of the multilayer coating design can be done byapplying one of the well known optical thin film calculation andoptimization techniques. In most cases these techniques are based onminimizing a merit function which measures the distance of the spectralcharacteristics of the actual thin film design to the targetcharacteristics. Within a slight modification of the standard processthe merit function can be modified in such a way that it comprises aterm which is addressing the distance of the square of the electricalfield to a desired value, which equals in the present case the maximumvalue of the standing wave.

In order to correctly determine the proper coating thicknessdistribution and/or index distribution, the incident angle of theexcitation light, the polarization and the refractive indices of thecover medium, of the coating material and of substrate must be takeninto account.

Please note that with such an optimization method it is as well possibleto design thin film stacks which meet the requirements of maximum fieldat the location of the areal fluorescent samples 23, 23′ for excitationsat two or more excitation wavelengths with the same multilayer coating.With the present invention it is therefore not necessary to compromisethicknesses to match the desired wavelengths.

The sample substrate 41 is designed to work with any fluorescenceimaging system, for example like that shown in FIG. 1. The samplesubstrate 41 according to the present invention is constructed tomaximize fluorescent emission and collection without having to increasethe power of the laser beam and without having to change the objectiveor other optics in the system other than the substrate 41 itself.

As an example a multilayer coating is given comprising 22 layers. Thecoating layer material, layer thickness distribution as well as theindex of refraction is listed in Table 1. For this example eachindividual layer thickness is designed using Nb₂O₅ (n=2.3) as materialwith a high optical refractive index and SiO₂ (n=1.48) as a materialwith a low optical refractive index. Other coating materials can beused. Multilayer coatings comprising more than two materials can be usedas well. In the present example the design is optimized for anexcitation wavelength of 532 nm.

FIG. 3 shows schematically the resulting normalized square of theelectrical field amplitude as a function of distance from the base 43.Shown as well is the distribution of the index of refraction as afunction of the distance from the base 43. As can be seen in FIG. 3 thesquare of the electrical field within the interference reflector 45 isoscillating from zero to a local maximum value and back to zero. Theenvelope of the local maxima within the interference reflector isincreasing with distance to the base. It is therefore not possible toidentify a reflecting surface. Since the excitation light penetratesseveral wavelenths into the interference reflection, definition of apenetration depth is not useful for defining a reflection surface.

As seen in FIG. 3, in case the proper coating thickness is provided, theareal fluorescent samples 23, 23′ is located at or near an antinode ofthe standing wave which is established above the interference reflector45 when the stimulating beam is reflected. The location of the arealfluorescent samples is especially indicated with an arrow as well aswith the broken line. With maximum excitation, maximum fluorescenceoccurs. Even if the coating thickness is not exactly correct for theexcitation wavelength, if the intensity is only 90% or 95% of the peakintensity, the fluorescence signal will still be significantly improvedover prior art sample substrates. Variations from the ideal thicknesscan occur due sample-to-sample variation and coating variations.

Further, if two or more different excitation wavelengths are to be usedwith the same substrate sample, an optimization strategy of themultilayer coating can be chosen to reach maximum field at the locationof the areal fluorescent samples for all excitation wavelengthsrequired. Thus, avoiding the usage of any undesired locations of lowerelectrical field for any required excitation wavelength.

The fluorescence imaging system of FIG. 1 could have one or more lightsources providing multiple fluorescence excitation wavelengths, eithersimultaneously or selectably, for different fluorescent sampleconstituents. The nominal optical thickness distribution of themultilayer coating needs and can then be optimized for each of thedifferent excitation wavelengths in parallel for the same multilayercoating.

This is illustrated in more detail in the following example:

Here for the two excitation wavelengths Cy5 at 633 nm and Cy3 at 532 nmthe sample substrate should provide enhanced electromagnetic field onthe physical surface. In order to achieve this we proceed according tothe following steps:

-   -   Designing of a first layer system, reflecting a first wavelength        range (620 nm-650 nm) and transmitting a second wavelength range        (520 nm-550 nm), thereby taking care that the outermost layer is        an SiO₂ layer.    -   Adjusting the outermost layer in such a way, that the square        amplitude electromagnetic field for the first excitation        wavelength (633 nm) on the physical surface of the layer system        is almost four times as high as the square of the amplitude        electromagnetic field of the incoming propagating plane wave.        With this the electromagnetic field conditions for the first        excitation wavelength are optimum on the surface. The layer        thicknesses for this first layer system are now fixed.    -   Placing an intermediate layer system between substrate and        adjusting the layer thicknesses in order to reflect the second        wavelength range.    -   Adjusting the thickness of the layer of the intermediate        layer system which is adjacent to the first layer system in such        a way, that the square amplitude electromagnetic field for the        second excitation wavelength (532 nm) on the physical surface of        the layer system is almost four times as high as the square of        the amplitude electromagnetic field of the incoming propagating        plane wave.

With this the electromagnetic field conditions for the second excitationwavelength as well as for the first excitation wavelength are optimum onthe surface.

Table two shows one example of such a sample substrate optimized forenhancement for the two excitation wavelengths 532 nm and 633 nm.

Because of this possibility it can be even in the case of the existanceof a well defined reflective surface (for example a metallic surface) asdescribed by Kain et al of advantage to skip the quarterwave conditionand to use a dielectric multilayer coating stack and the optimizationprocess as described above to realize maximum fields at the location ofthe areal fluorescent samples for one more than one excitationwavelength.

This is illustrated in more detail in the following example:

The first excitation wavelength (633 nm) is reflected by the dielectricinterference layers whereas the second excitation wavelength isreflected by the silver layer. The outermost layer of this coating layersystem can be used to adjust the electromagnetic field of the firstexcitation wavelength on the physical surface by adjusting the layerthickness. The first layer on the silver coating can be used to adjustthe electromagnetic field of the second excitation wavelength byadjusting the layer thickness. Table 3 gives an example of a thicknessdistribution of such a layer system.

In our examples we used always SiO₂ as outermost layer. This is thestandard material for applying linker chemistry. Linker chemistries arequite sensitive to the materials used on the physical surface of thesubstrate sample. Therefore different linker chemistries might requireoutermost layers different from SiO₂. It is clear that the concept ofthe invention should not be limited to the use of SiO₂ as outermostlayers. In practical examples we already used TiO₂ and/or Nb₂O₅. Anyother practical optical coating material could be used as well.Additionally, in case the fluorescent emission is be detected from thebackside of the substrate, the approach of the present invention can beused due to the absence of any metal layer incorporated. In this casethe multilayer coating can be in addition optimized to effectivelytransmit the fluorescent light.

1. A sample substrate adapted for use with fluorescence excitation lightwith a first wavelength and a second wavelength, comprising a base and areflector, the reflector comprising a reflecting multilayer interferencecoating comprising a first layer system including multiple layers and anintermediate layer system including multiple layers, wherein the firstlayer system reflects the excitation light with the first wavelength andtransmits the excitation light with the second wavelength, andthicknesses of the layers of the first layer system ensure that anyfluorescent sample material deposited on top of said multilayerinterference coating is located near an antinode of a standing waveformed by the excitation light with the first wavelength incident onsaid substrate, and wherein the intermediate layer system reflects theexcitation light with the second wavelength, and a thickness of at leastone layer of the intermediate layer system ensures that the samplematerial deposited on top of said multilayer interference coating islocated near an antinode of a standing wave formed by the excitationlight with the second wavelength incident on said substrate, and furtherwherein the reflecting multilayer interference coating causes theantinodes to be located at substantially the same position.
 2. Thesample substrate according to claim 1, wherein said first wavelength iswithin the wavelength range of 532 nm to 548 nm and said secondwavelength is around 633 nm.
 3. The sample substrate according to claim1, wherein the reflector is metal-free.
 4. The sample substrateaccording to claim 1, wherein the base provides a metallic reflectingsurface.
 5. The sample substrate according to claim 1, wherein anoutermost layer of the reflecting multilayer interference coating is aSiO₂ layer.
 6. The substrate of claim 1, wherein the intermediate layersystem comprises a metal layer disposed on the base and a dielectriclayer disposed on the metal layer.
 7. The substrate of claim 6, whereinthe metal layer is one metal of the group comprising silver, gold,aluminum, chromium, platinum and any alloys of these metals.
 8. Thesubstrate of claim 1, wherein the multilayer interference coatingcomprises dielectric layers having alternately relatively higher andlower refractive indices such that an outermost layer is one of thelayers having a relatively lower refractive index.
 9. The substrate ofclaim 8, wherein the layers comprise at least one of Nb2O5, SiO2,aluminum oxide, magnesium oxide; oxides of the groups VIb, Vb, IVb,scandium, yttrium, calcium, strontium, zinc, iron, indium, tin, cerium,or holmium; oxides of mixtures or alloys of scandium, yttrium, calcium,strontium, zinc, iron, indium, tin, cerium, or holmium; and oxynitridesof Ti, Ta, Zr, Si, Hf, or Al; fluorides of magnesium, barium, strontium,calcium, rare earths, and lead.
 10. The substrate of claim 8, whereinthe layers comprise Nb2O5 and SiO2.
 11. The substrate of claim 1,further comprising a linking coating disposed on an outermost layer ofthe reflector.
 12. The substrate of claim 11, wherein the linkingcoating layer is biologically active.
 13. The substrate of claim 1,wherein the base is rigid.
 14. The substrate of claim 1, wherein thebase comprises one of glass, plastic, metal, or semiconductor material.15. A fluorescence imaging system comprising a sample substrateaccording to claim 1 and a light source directed to the sample materialon said sample substrate, said light including the fluorescenceexcitation wavelength and being particular to a specified fluorescentconstituent of said sample material.
 16. A laser scanner comprising afluorescence imaging system according to claim 15.